The development of inactivated vaccines or vaccines containing purified antigens is increasingly important since it makes it possible to avoid adverse side effects in the individual treated. However, the improvement in the quality of antigens occurs to the detriment of the immunogenic nature of said antigens. This is why they are combined with adjuvants of immunity.
Adjuvants of immunity are products which increase immune system reactions, when they are administered in the presence of antigens of viral, bacterial or synthetic origin. They bring about the massive appearance of macrophages at the injection site, and then in the lymph nodes, increase the production of specific immunoglobulins, the antibodies, and stimulate numerous cells involved in immune defense mechanisms.
These adjuvants are of diverse natures. They may, for example, consist of emulsions which are in the form of water-in-oil W/O, or oil-in-water O/W, or water-in-oil-in-water W/O/W, or oil-in-water-in-oil O/W/O emulsions.
Freund's adjuvants are very effective; they result from the combination of a mineral oil and of a mannitol ester optionally containing a killed mycobacterium. Vaccines prepared by mixing, in equal parts, a Freund's adjuvant with an aqueous antigenic medium still remain the references throughout the world for laboratory studies. They are in the form of water-in-oil W/O emulsions, i.e. of emulsions for which the continuous phase is constituted of an oil or a mixture of oils, and the dispersed phase is an aqueous phase that may comprise solubilizing excipients such as, for example, glycerol or dimethyl sulfoxide. The active ingredient is generally located in the aqueous phase, which is commonly a buffered saline solution. This phase is in the form of drops separated from one another by an oily film. This formulation makes it possible to obtain amplified biological responses sustained over time.
However, emulsions of this W/O type are generally very viscous and are therefore difficult to inject. They often require the use of syringes with a large needle diameter and cause pain during the injection and trauma at the injection site.
The aqueous phase content by mass of injectable W/O emulsions is around 30% to 40% for 100% of the mass of the emulsion. The maximum content by mass encountered is 30%. However, this limit constitutes an impairment in particular to the development of polyvalent vaccines, in which several antigens are combined and for which it would be preferable to obtain W/O emulsions having aqueous phase contents of greater than 50% by mass.
However, in a W/O emulsion with a low aqueous phase content, up to approximately 20% by mass, the viscosity of the emulsion is very close to the viscosity of the oil. Increasing the proportion by mass of aqueous phase increases its viscosity. Thus, an emulsion containing 20% by mass of water, having a viscosity of 100 mPa·s, measured using a Brookfield LVT viscometer fitted with a No. 2 spindle revolving at a speed of 60 revolutions per minute, changes into a cream that is very difficult to inject when the water content is increased up to 50% by mass.
Some commercial oily adjuvants which are in the form of W/O emulsions, such as Montanide™ ISA 70, make it possible to obtain injectable W/O emulsions containing approximately 30% by weight of aqueous phase and 70% by weight of oily phase and having viscosities of the order of 50 to 100 mPa·s measured using a Brookfield LVT viscometer fitted with a No. 2 spindle revolving at a speed of 60 revolutions per minute. Other adjuvants, such as Montanide™ ISA 50V2, make it possible to obtain injectable W/O emulsions containing approximately 50% by weight of aqueous phase and 50% by weight of oily phase and having viscosities of less than 250 mPa·s, measured using a Brookfield LVT viscometer fitted with a No. 2 spindle revolving at a speed of 60 revolutions per minute.
The international patent application published under the number WO 99/20305 discloses the use of mannitol oleate-based surfactants with mineral oils such as Marcol™ 52 for preparing “fluid” emulsions having viscosities of the order of 500 mPa·s measured using a Brookfield LVT viscometer fitted with a No. 2 spindle revolving at a speed of 60 revolutions per minute.
The notion of a fluid emulsion depends largely on the field of application. For injectable emulsions, the upper viscosity limit for a fluid emulsion is defined with respect to the viscosity of a reference W/O emulsion which contains 50% by mass of aqueous phase and 50% by mass of incomplete Freund's adjuvant (IFA); this viscosity, measured using a Brookfield LVT viscometer fitted with a No. 3 spindle revolving at a speed of 30 revolutions per minute, is of the order of approximately 2000 mPa·s. This emulsion is considered to be very viscous.
A W/O emulsion will be said to be fluid if its viscosity is less than a quarter of that of this reference emulsion, i.e. less than 500 mPa·s at 25° C., measured using a Brookfield LVT viscometer fitted with a No. 2 spindle and revolving at a speed of 30 revolutions per minute.
However, fluid emulsions are generally less stable than more viscous emulsions, since phase separations at ambient temperature are observed only a few days after preparation of said emulsions.
Aqueous phase thickening polymers in the form of powders exist, such as acrylic acid homopolymers in the sodium forms thereof or copolymers based on acrylic acid and its esters. Mention will, for example, be made of the polymers sold by the company Noveon under the trade mark Carbopol™ and Pemulen™. They are described in particular in U.S. Pat. No. 5,373,044 and U.S. Pat. No. 2,798,053 and in European patent EP 0 301 532. These polymers were initially developed as thickeners for thickening formulations essentially intended for cosmetic applications, and which have been described and used for many years. These polymers are obtained from a monomer such as, for example, acrylic acid, methacrylic acid, acrylic acid esters or methacrylic acid esters in solution in an organic solvent phase.
During the polymerization reaction, obtained by adding various catalysts under specific temperature and pressure conditions, the polymer becomes insoluble in the initial solvent phase and precipitates at the bottom of the reactor. This process is called “precipitation polymerization”.
The polymers thus obtained, the most well-known grades of which are the Carbopols©, are very effective thickeners that are very widely used in the cosmetics industry. These polymers are also used for varied pharmaceutical applications:                preparation of delayed-release-effect polymer matrices,        preparation of polymer gels, and complexation of said polymers with proteins,        preparation of polymer films creating protection during contacting with biological fluids,        preparation of bioadhesive formulations providing greater persistence during application to the mucous membranes,        preparation of vaccine adjuvants.        
The use as a vaccine adjuvant, with final polymer contents by mass of the order of “one percent”, is in the form of a readily injectable, translucent, fluid vaccine.
The adjuvant properties of synthetic substances are very closely linked to their physical forms at the time of administration. Thus, the size of particles used to trigger a large adjuvant effect is a major parameter which will control the possibility of picking up by the immunocompetent cells. Furthermore, depending on the size of the particles, it will also be possible to orient the response toward antibody production (humoral response) or rather for stimulating specific immune system cells (cellular response). The synthesis of microspheres of various polymers in a solvent phase in the form of dispersions is widely described in the literature with the possibility of encapsulation of a biologically active agent (which may be an antigen in the case of vaccines) or being used as an adsorption support for carrying and presenting an active agent bound to the external surface of the microspheres by more or less strong interactions.
The controlling of the size of the adjuvant particles is a constant challenge since it must be accompanied by stability of these adjuvant particles over time. It is important in particular to avoid aggregations during storage or phase separations. The controlling of the size must also be accompanied by stability of the composition, after injection into the living being, in particular owing to stresses linked to variation in pH, to the presence of enzymes, to temperature variations.